The overall goal of this AREA renewal application is focused on the development of new microchip approaches to study cell-to-cell-interactions. Cell-to-cell communication is an important component in understanding cell function. A better understanding of these interactions could lead to important advances in the medical and neuroscience fields, but a lack of technology currently limits the ability to study the interaction between cell lines at the molecular level. In aim #1, we propose to develop a new and innovative chip-to-chip approach that can be used to study the interaction between two adherent cells, PC 12 cells and microglia cells. While there have been reports of using highly integrated microchip devices for analyzing cells on-chip, they are relatively low throughput and if any part of the chip fails the entire experiment is sacrificed. To address these issues, we propose in aim #1 the development of a new approach, a polystyrene (PS)-based chip-to-chip device in which a cell culture/immobilization device is isolated from the analysis device. This more modular approach will enable the numerous advantages of doing cell culture in microchip devices and, when analysis is desired by microchip electrophoresis and electrochemical detection, our recently reported ability to encapsulate both tubing and electrodes in PS devices will lead to a low dead volume connection to direct flow from the cell chip to the analysis chip. In addition, we propose a new type of device that interfaces a bilayer PDMS-based microchip with an embedded fused-silica capillary to enhance the separation capabilities. In essence, this approach will bridge the fundamental advantages of PDMS devices (including the ability to incorporate valves and electrochemical detection) with widely used fused-silica capillary for more efficient separations. In specific aim #2, we propose a new type of planar membrane device that can be used to study the interaction between a flowing cell line (red blood cells) and adherent cells (endothelial cells). The focus of this aim is detection of one of the key chemical messengers, nitric oxide (NO), an analytical challenging molecule that has a very short half-life in vivo. The development of a new and innovative system capable of electrochemically monitoring NO release from immobilized endothelial cells as a response to RBC derived ATP release within seconds is essential to a better understanding of cellular signaling at the molecular level and could lead to further knowledge in the cardiovascular field. In this aim, we propose a novel PS-based device that uses a pillar array to create a planar membrane that will allow small molecules such as NO to pass through the pores created by the array into an adjacent channel (collector channel), where they will be electrochemically detected using our recently reported Pt-black electrodes. The planar membrane approach will enable visualization of all cells lines as well as allowing the collector channel and the detection electrodes to be aligned in very close proximity to the membrane (but away from the channel containing cells), which is essential for detecting a short-lived molecule such as NO.